The overall goal of this research project is to discover lead compounds that can be developed into therapeutics for treatment of Chagas disease. This life-threatening disease results from infection with the protozoan pathogen Trypanosoma cruzi (T. cruzi). It is endemic in South and Central America, with >16 million people chronically infected and approximately 14,000 deaths per year. More than 100,000 USA citizens are believed to be infected. Current treatment options are inadequate to cure this infection, thus research is needed to discover better chemotherapeutics. The project will focus on making inhibitors to a key enzyme involved in sterol biosynthesis in T. cruzi, sterol 14-demethylase (Tc14DM). This enzyme, the target of azole antifungal drugs, is a well established antimicrobial target. We hypothesize that new compounds can be synthesized with optimal anti-T. cruzi activity and these will be sufficiently active to cure animals (and humans) with chronic T. cruzi infection. We provide preliminary data on two new chemical classes that bind the Tc14DM and that have potent activity against T. cruzi cultures. The specific aims are: 1) Synthesize analogs of tipifarnib as potential T. cruzi chemotherapeutics. Tipifarnib is an imidazole- containing compound under development for cancer therapy and has excellent pharmacokinetic properties. 2) Synthesize a series of disubstituted imidazoles as potential T. cruzi chemotherapeutics. Preliminary data shows that this series has potent anti-T. cruzi activity when given orally to infected mice. 3) Perform molecular modeling and structure determination of Tc14DM. A molecular model based on a prokaryotic CYP51 structure will initially be utilized to guide structure-based drug design. Concomitantly, a crystal structure of the enzyme with bound inhibitors will be pursued to help guide drug design. 4) Develop a competition binding assay for Tc14DM. The proposed fluorescence polarization assay will assist moderate- to high-throughput screening of compounds. 5) Test compounds for efficacy, pharmacokinetics, and toxicity. A series of in vitro screens will be employed followed by testing selected compounds in mouse models.